Viral contamination in biological samples is a problem in a number of areas of medicine including blood transfusion and organ transplantation, and vaccine and drug production. Viruses can be detected in biological samples using a number of different tests that detect the presence of viral antigens, host antibodies to viral antigens or viral nucleic acids.
When testing a biological sample for contamination by a virus or confirming that a biological sample is free from a virus, a problem exists with interpreting a negative result. Without appropriate controls, it is not possible to determine whether an absence of contaminating virus being detected is as a result of the failure of the assay, or as a result of the absence of any contaminating virus in the biological sample. If the negative result can be attributed to the former reason, the failure of the assay could have occurred at any stage. For example, in a nucleic acid assay, the failure may have occurred during nucleic acid extraction, handling, amplification or detection steps. Generally, four controls are used in PCR based methods for the detection of viral nucleic acids. The first control is an internal positive control for the nucleic acid extraction step. The second control is for the detection of the PCR products. The third control is for the amplification step. Finally, the fourth control is a no template control to detect contamination during the assay. Similar controls are used in assays to detect viral polypeptides.
The present invention relates to the use of a second virus as an exogenous internal positive.
The concept of using an exogenous virus as an internal positive control for diagnostic purposes is known in the art. For example, Mairhofer et al. (qPCR 2007 Symposium & Exhibition & Workshop 3rd International qPCR Symposium, page 28. ISBN-13 978-3-00-020385-5) describes the use of Tomato Mosaic Virus (ToMV) as an internal positive control in an assay for the detection of influenza A virus in a biological sample from a subject infected with influenza A. This system, however, has several disadvantages. In particular, ToMV did not work as an internal positive control for the detection of Norovirus I from clinical specimens and its use as an internal positive control is therefore limited.
A further disadvantage of the system described by Mairhofer et al. is that the control virus (ToMV) and the test virus (influenza A) are different types of virus. ToMV is a non-enveloped virus with a +ssRNA genome. On the other hand, influenza A is an enveloped virus with a −ssRNA genome. This is likely to lead to a lack of reliability of the positive control. For example, if extraction, amplification and detection steps are optimised for the positive control virus it is possible that the conditions in any one of the extraction, amplification and detection steps would not be suitable for the contaminating virus. Thus, a negative result for the detection of the contaminating virus when the assay is positive for the detection of the control virus could represent a failure of the extraction, amplification or detection step to work for the contaminating virus rather than an absence of contamination in the biological sample. Indeed, Mairhoffer et al. reported at the 2007 qPCR symposium that ToMV and Norovirus nucleic acids can not both be detected in a same sample known to contain both viruses when the nucleic acids are extracted under the same conditions.
A yet further disadvantage of the use of ToMV is that the virus is commonly found in all solenaceous plant (for example tobacco, potato and tomato). Thus, contamination of the assay with the positive control after nucleic acid extraction is possible from commonly found plant materials. If a sample were to be contaminated after the nucleic acid extraction step, a positive result for the detection of the control virus ToMV could result even if the extraction step failed.
There therefore remains a need for a suitable internal positive control for nucleic acid extraction and detection assays. To our knowledge, the use of exogenous viruses as an internal positive control (IPC) in vaccine manufacturing has not been described previously.